Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation.

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer.

Covalent amino acid modification significantly expands protein functional capability in regulating biological processes. Tyrosine residues can undergo phosphorylation, sulfation, adenylation, halogenation, and nitration. These posttranslational modifications (PTMs) result from the actions of specific enzymes: tyrosine kinases, tyrosyl-protein sulfotransferase(s), adenylate transferase(s), oxidoreductases, peroxidases, and metal-heme containing proteins. Whereas phosphorylation, sulfation, and adenylation modify the hydroxyl group of tyrosine, tyrosine halogenation and nitration target the adjacent carbon residues. Because aberrant tyrosine nitration has been associated with human disorders and with animal models of disease, we have created an updated and curated database of 908 human nitrated proteins. We have also analyzed this new resource to provide insight into the role of tyrosine nitration in cancer biology, an area that has not previously been considered in detail. Unexpectedly, we have found that 879 of the 1971 known sites of tyrosine nitration are also sites of phosphorylation suggesting an extensive role for nitration in cell signaling. Overall, the review offers several forward-looking opportunities for future research and new perspectives for understanding the role of tyrosine nitration in cancer biology.

Revisiting the role of 3-nitrotyrosine residues in the formation of alpha-synuclein oligomers and fibrils.

Nitration of tyrosine residues in alpha-synuclein (a-syn) has been detected in different synucleinopathies, including Parkinson's disease. The potential role of 3-nitrotyrosine formation in a-syn, as an oxidative post-translational modification, is still elusive. In this work, we generated well-characterized tyrosine nitrated a-syn monomers and studied their capability to form oligomers and fibrils. We constructed tyrosine to phenylalanine mutants, containing a single tyrosine residue, a-syn mutant Y(125/133/136)F and Y(39/125/133)F) and assessed the impact in a-syn biophysical properties. Nitrated wild-type a-syn and the Y-F mutants, with one 3-nitrotyrosine residue in either the protein's N-terminal or C-terminal region, showed inhibition of fibril formation but retained the capacity of oligomer formation. The inhibition of a-syn fibrillation occurs even when an important amount of unmodified a-syn is still present. We characterized oligomers from both nitrated and non-nitrated forms of the wild-type protein and the mutant forms obtained. Our results indicate that the formation of 3-nitrotyrosine in a-syn could induce an off-pathway oligomer formation which may have an important impact in the development of synucleinopathies.

CEACAM6 as a Novel Therapeutic Target to Boost HO-1-mediated Antioxidant Defense in COPD.

Rationale: Tobacco smoking and air pollution are primary causes of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop COPD. The mechanisms underlying the defense against nitrosative/oxidative stress in nonsusceptible smokers to COPD remain largely unresolved. Objectives: To investigate the defense mechanisms against nitrosative/oxidative stress that possibly prevent COPD development or progression. Methods: Four cohorts were investigated: 1) sputum samples (healthy, n = 4; COPD, n = 37), 2) lung tissue samples (healthy, n = 13; smokers without COPD, n = 10; smoker+COPD, n = 17), 3) pulmonary lobectomy tissue samples (no/mild emphysema, n = 6), and 4) blood samples (healthy, n = 6; COPD, n = 18). We screened 3-nitrotyrosine (3-NT) levels, as indication of nitrosative/oxidative stress, in human samples. We established a novel in vitro model of a cigarette smoke extract (CSE)-resistant cell line and studied 3-NT formation, antioxidant capacity, and transcriptomic profiles. Results were validated in lung tissue, isolated primary cells, and an ex vivo model using adeno-associated virus-mediated gene transduction and human precision-cut lung slices. Measurements and Main Results: 3-NT levels correlate with COPD severity of patients. In CSE-resistant cells, nitrosative/oxidative stress upon CSE treatment was attenuated, paralleled by profound upregulation of heme oxygenase-1 (HO-1). We identified carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a negative regulator of HO-1-mediated nitrosative/oxidative stress defense in human alveolar type 2 epithelial cells (hAEC2s). Consistently, inhibition of HO-1 activity in hAEC2s increased the susceptibility toward CSE-induced damage. Epithelium-specific CEACAM6 overexpression increased nitrosative/oxidative stress and cell death in human precision-cut lung slices on CSE treatment. Conclusions: CEACAM6 expression determines the hAEC2 sensitivity to nitrosative/oxidative stress triggering emphysema development/progression in susceptible smokers.

Development and application of novel ELISA-based analytical tools for assessing nitroxidative distress biomarkers in ischemic stroke: implications for improved diagnosis and clinical management.

Ischemic cerebrovascular accident (iCVA) is a public health issue, whose subjacent events involve the development of nitroxidative distress. Identifying biomarkers that assist in the diagnosis of this disease has clinically relevant implications. The aim of this study was to develop an analytic tool for measuring nitroxidative distress biomarkers, intended for application in clinical practice to enhance patient healthcare. Three enzyme linked immunosorbent assays (ELISA) were developed, with different detection objectives. One of them, in a sandwich format, quantifies the amount of fibrinogen in human plasma, an important glycoprotein involved in the blood coagulation process, contributing to thrombus formation and thereby participating in the mechanism of ischemic stroke. Another ELISA, also in a sandwich format, detects the presence of nitrotyrosine residues in fibrinogen from human plasma, a nitroxidative posttranslational modification resulting from the attack of peroxynitrite by-products on tyrosine residues present in proteins. The third one, in inhibition format, determines human plasma nitrotyrosine total content and was used to analyze human plasma samples from control and iCVA patients. Those two groups of plasma samples were analyzed using inhibition ELISA, revealing statistically significant differences in their nitrotyrosine content and molar ratios of nitrotyrosine to fibrinogen, which were higher in the iCVA group. This study provides evidence that nitroxidative distress occurs in ischemic stroke, as indicated by the detection of the biomarker nitrotyrosine. This finding supports other studies that also identified nitrotyrosine in ischemic stroke, through several different methods. This specific ELISA method is applicable for the rapid analysis of clinical samples, making it a potential clinical tool for assessing iCVA patients.

Hyperbaric Oxygen Reduces Oxidative Stress Impairment and DNA Damage and Simultaneously Increases HIF-1α in Ischemia-Reperfusion Acute Kidney Injury.

The central exacerbating factor in the pathophysiology of ischemic-reperfusion acute kidney injury (AKI) is oxidative stress. Lipid peroxidation and DNA damage in ischemia are accompanied by the formation of 3-nitrotyrosine, a biomarker for oxidative damage. DNA double-strand breaks (DSBs) may also be a result of postischemic AKI. γH2AX(S139) histone has been identified as a potentially useful biomarker of DNA DSBs. On the other hand, hypoxia-inducible factor (HIF) is the "master switch" for hypoxic adaptation in cells and tissues. The aim of this research was to evaluate the influence of hyperbaric oxygen (HBO) preconditioning on antioxidant capacity estimated by FRAP (ferric reducing antioxidant power) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay, as well as on oxidative stress parameter 3-nitrotyrosine, and to assess its effects on γH2AX(S139), HIF-1α, and nuclear factor-κB (NF-κB) expression, in an experimental model of postischemic AKI induced in spontaneously hypertensive rats. The animals were divided randomly into three experimental groups: sham-operated rats (SHAM, n = 6), rats with induced postischemic AKI (AKI, n = 6), and group exposed to HBO preconditioning before AKI induction (AKI + HBO, n = 6). A significant improvement in the estimated glomerular filtration rate, eGFR, in AKI + HBO group (p < 0.05 vs. AKI group) was accompanied with a significant increase in plasma antioxidant capacity estimated by FRAP (p < 0.05 vs. SHAM group) and a reduced immunohistochemical expression of 3-nitrotyrosine and γH2AX(S139). Also, HBO pretreatment significantly increased HIF-1α expression (p < 0.001 vs. AKI group), estimated by Western blot and immunohistochemical analysis in kidney tissue, and decreased immunohistochemical NF-κB renal expression (p < 0.01). Taking all of these results together, we may conclude that HBO preconditioning has beneficial effects on acute kidney injury induced in spontaneously hypertensive rats.

Age-Related COVID-19 Influence on Male Fertility.

The impact of coronavirus on the reproductive health of men attracts the special attention of many researchers. While studies suggest changes in sperm parameters and the possibility of testicular inflammation, further studies are needed to elucidate any potential age-related changes in these findings, which is the purpose of the present study. The semen quality parameters, cytokine concentration, and markers of the pro- and antioxidant system were assessed in 60 men five to seven months after the coronavirus infection and in 77 controls (without a history of coronavirus infection). Additionally, participants were divided into two age groups: less than 35 years and 35 years or older. Notably increased round cell count in ejaculate and reduced sperm hyaluronan binding ability were observed among post-infection patients younger than 35 years. In the same group, a decline in seminal plasma zinc levels and nitrotyrosine in the cell fraction was found. In men over 35 years of age, Coronavirus Disease 2019 (COVID-19) led to increased sperm DNA fragmentation, a decrease in the total antioxidant capacity, and an elevation in the levels of interleukin-1ß and interleukin-10. The concentration of interleukin-1ß decreased over time following recovery in all affected patients. The data obtained suggest the potential adverse impact of the coronavirus infection on male reproductive health; however, these effects appear to be age-dependent.

Effects of Normobaric Hypoxia and Adrenergic Blockade over 72 h on Cardiac Function in Rats.

In rats, acute normobaric hypoxia depressed left ventricular (LV) inotropic function. After 24 h of hypoxic exposure, a slight recovery of LV function occurred. We speculated that prolonged hypoxia (72 h) would induce acclimatization and, hence, recovery of LV function. Moreover, we investigated biomarkers of nitrosative stress and apoptosis as possible causes of hypoxic LV depression. To elucidate the role of hypoxic sympathetic activation, we studied whether adrenergic blockade would further deteriorate the general state of the animals and their cardiac function. Ninety-four rats were exposed over 72 h either to normal room air (N) or to normobaric hypoxia (H). The rodents received infusion (0.1 mL/h) with 0.9% NaCl or with different adrenergic blockers. Despite clear signs of acclimatization to hypoxia, the LV depression continued persistently after 72 h of hypoxia. Immunohistochemical analyses revealed significant increases in markers of nitrosative stress, adenosine triphosphate deficiency and apoptosis in the myocardium, which could provide a possible explanation for the absence of LV function recovery. Adrenergic blockade had a slightly deteriorative effect on the hypoxic LV function compared to the hypoxic group with maintained sympathetic efficacy. These findings show that hypoxic sympathetic activation compensates, at least partially, for the compromised function in hypoxic conditions, therefore emphasizing its importance for hypoxia adaptation.

Cross-linking and modification of fibronectin by peroxynitrous acid: Mapping and quantification of damage provides a new model for domain interactions.

Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where it is exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitrotyrosine, di-tyrosine, and 6-nitrotryptophan. We have shown previously that human plasma FN is readily modified by ONOOH, but the extent and location of modifications, and the role of FN structure (compact versus extended) in determining these factors is poorly understood. Here, we provide a detailed LC-MS analysis of ONOOH-induced FN modifications, including the extent of their formation and the sites of intramolecular and intermolecular cross-links, including Tyr-Tyr, Trp-Trp, and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of ECM modification and turnover in the many pathologies associated with inflammation-associated fibrosis.

Dynamics of nitration during dark-induced leaf senescence in Arabidopsis reveals proteins modified by tryptophan nitration.

Nitric oxide (NO) is a critical molecule that links plant development with stress responses. Herein, new insights into the role of NO metabolism during leaf senescence in Arabidopsis are presented. A gradual decrease in NO emission accompanied dark-induced leaf senescence (DILS), and a transient wave of peroxynitrite (ONOO-) formation was detected by day 3 of DILS. The boosted ONOO- did not promote tryptophan (Trp) nitration, while the pool of 6-nitroTrp-containing proteins was depleted as senescence progressed. Immunoprecipitation combined with mass spectrometry was used to identify 63 and 4 characteristic 6-nitroTrp-containing proteins in control and individually darkened leaves, respectively. The potential in vivo targets of Trp nitration were mainly related to protein biosynthesis and carbohydrate metabolism. In contrast, nitration of tyrosine-containing proteins was intensified 2-fold on day 3 of DILS. Also, nitrative modification of RNA and DNA increased significantly on days 3 and 7 of DILS, respectively. Taken together, ONOO- can be considered a novel pro-senescence regulator that fine-tunes the redox environment for selective bio-target nitration. Thus, DILS-triggered nitrative changes at RNA and protein levels promote developmental shifts during the plant's lifespan and temporal adjustment in plant metabolism under suboptimal environmental conditions.

Photo-tuneable protein nitration by sensitiser tris(bipyridine)-Ruthenium(II) chloride complex.

Post-translational modifications (PTMs) of proteins are a diverse source of variability that impacts on their functions, localisation, regulation, and lifetime. However, one of the main pitfalls in their study is that they appear in rather low frequencies and/or are only transiently observed. To overcome this issue and ease the study in vitro of stress-related protein PTMs, several methods have been proposed to model stress conditions and chemically introduce them. These techniques employ the combination of peroxides with transition metal ions or haem-containing proteins, as well as other possibilities such as peroxy radicals or UV radiation. However, their control, reproducibility and undesired secondary reactions that reduce the process yield are often a matter of concern. Here we introduce a photo-tuneable method that selectively targets nitration of aromatic residues. We initially present the adaptation of an oxidation method based on the photosensitiser tris(2,2'-bipyridine)-Ruthenium(II) chloride complex and ammonium persulfate, in which we employ an alternative radical neutralisation/trapping pathway that uses nitrite ions for the nitration of free l-Tyrosine and L-Tryptophan amino acids. After analysing the effect of several factors, we report the application of the photo-tuneable protein nitration (PTPN) method to four different model proteins in which we evaluate the nitration and oxidation of residues in each case. A mass spectrometry label-free quantitation of Tyr and Trp nitration is also described in order to compare the degree of modification and the accessibility of these residues. The method described could be employed to scale up the production of proteins with a selected range of oxidative PTMs for their characterisation, the assessment of their pathophysiological roles, and the development of detection and quantification methods to validate these PTMs as novel biomarkers associated with oxidative stress-related pathologies, such as in cardiovascular or neurodegenerative diseases.

Heterogeneous nitration reaction of BSA protein with urban air: improvements in experimental methodology.

Gas-phase ozone (O3) and nitrogen dioxide (NO2) can react with environmentally exposed proteins to induce chemical modifications such as the formation of nitrotyrosine (NTyr). Certain proteins with these modifications have also been shown to promote adverse health effects and can trigger an immune response. It is hypothesized that proteinaceous material suspended in the atmosphere as particulate matter, e.g., embedded in pollen, can undergo heterogenous reactions to produce chemically modified proteins that impact human health, especially in urban areas. To investigate the protein modification process under ambient outdoor reaction conditions, bovine serum albumin (BSA) protein samples were loaded onto filters and exposed to urban air in Denver, Colorado (USA). Losses and measurement artifacts were measured independently to calculate nitration effects on the protein via high-performance liquid chromatography and to support the experimental methodology. O3 loss from inlet lines using three commonly used particulate filters was quantified, showing a range of ambient O3 concentration losses from 3.2% for Kynar® (polyvinylidene fluoride) filters to > 60% for commonly used HEPA filters. Protein mass extraction efficiency was calculated as a function of filter material and protein mass using both native and nitrated BSA. Finally, we show examples of BSA samples nitrated by exposure to urban air as a proof-of-concept for future studies, highlighting the potential for atmospherically relevant NTyr formation. The methodology vetted here provides support for a wide variety of experimental efforts related to exposure of analytes to O3 and more broadly to an expanding field of protein modification in ambient air.

Serum fractalkine and 3-nitrotyrosine levels correlate with disease severity in Parkinson's disease: a pilot study.

Parkinson's disease (PD) and Parkinsonian syndromes; Progressive supranuclear palsy (PSP), and Multiple system atrophy (MSA) are debilitating neurodegenerative disorders. Fractalkine is a chemokine involved in neuroinflammation, whereas, 3-nitrotyrosine (3-NT) is a marker of early neurodegenerative cellular-damage. We measured Fractalkine and 3-NT levels in the serum of these patients to examine the neuroinflammation hypothesis and also to decipher the propensity of these biologics to be used as early (5 years from onset) biochemical markers in neurodegenerative Parkinsonism. The diagnoses of PD, PSP and MSA were performed as per the respective clinical criteria. 21 PD, 9 PSP and 8 MSA patients along with controls participated in this study. Serum concentrations of Fractalkine and 3-NT were measured by ELISA. Fractalkine levels were increased in PD, PSP and MSA cohorts in comparison with controls with p < 0.001, p < 0.05 and p < 0.05 respectively. Levels of 3-NT also showed elevation in PD (p < 0.01) vs. controls. However, Pearson plot showed that Fractalkine levels were high in the patients with unified Parkinson's disease rating scale (UPDRS) part III motor score of 1, meaning slight disability, but gradually dropped in patients with motor score of 4, which is a measure of severe motor disability. This negative correlation (- .565, p < .0.01) also accentuates the neuroprotectant/anti-inflammatory nature of Fractalkine in PD. Continuous rise of 3-NT in PD, positively correlating (.512, p < 0.05) with worsening motor symptoms points to deleterious consequences of nitrosative stress. To our knowledge, this is the first report providing evidence that serum Fractalkine and 3-NT have early diagnostic/prognostic significance as PD biomarkers.

Extended Prophylactic Effect of N-tert-Butyl-α-phenylnitron against Oxidative/Nitrosative Damage Caused by the DNA-Hypomethylating Drug 5-Azacytidine in the Rat Placenta.

Antioxidant N-tert-Butyl-α-phenylnitron (PBN) partly protected embryos from the negative effects of a DNA demethylating drug 5-azacytidine during pregnancy. Our aim was to investigate PBN's impact on the placenta. Fischer rat dams were treated on gestation days (GD) 12 and 13 by PBN (40 mg/kg), followed by 5azaC (5 mg/kg) after one hour. Global methylation was assessed by pyrosequencing. Numerical density was calculated from immunohistochemical expression in single cells for proliferating (PCNA), oxidative (oxoguanosine) and nitrosative (nitrotyrosine) activity. Results were compared with the PBN-treated and control rats. PBN-pretreatment significantly increased placental weight at GD15 and GD20, diminished by 5azaC, and diminished apoptosis in GD 20 placentas caused by 5azaC. Oxoguanosine expression in placentas of 5azaC-treated dams was especially high in the placental labyrinth on GD 15, while PBN-pretreatment lowered its expression on GD 15 and GD 20 in both the labyrinth and basal layer. 5azaC enhanced nitrotyrosine level in the labyrinth of both gestational stages, while PBN-pretreatment lowered it. We conclude that PBN exerted its prophylactic activity against DNA hypomethylating agent 5azaC in the placenta through free radical scavenging, especially in the labyrinthine part of the placenta until the last day of pregnancy.

Oxidative Stress Markers and Sperm DNA Fragmentation in Men Recovered from COVID-19.

SARS-CoV-2 negatively affects semen characteristics, impairs various biochemical processes in seminal fluid and within spermatogenic cells ultimately leading to male fertility decline. However, the distinct mechanisms, in particular, the role of oxidative stress on the consequences of coronavirus infection, have not been well investigated, which is the purpose of the present study. The standard semen parameters, its pro- and antioxidant system state, as well as the level of sperm DNA fragmentation, were assessed in 17 semen samples of men five months after the coronavirus infection and in 22 age-matched control patients. We determined that the DNA fragmentation rate negatively correlated with the period after coronavirus recovery, as well as seminal fluid superoxide dismutase activity and uric acid level. It was demonstrated that COVID-19 is not always associated with increased DNA fragmentation, allowing them to be considered as two independent factors. Thus, the most significant changes were noted in the samples of men after COVID-19 and abnormal TUNEL results: increased round cell number, decreased seminal fluid's nitrotyrosine level, and total antioxidant capacity and Zn, as well as an increased 8-hydroxy-2'-deoxyguanosine level within spermatozoa. The data obtained indicate that increased DNA fragmentation and diminished semen quality in men can be the result of an imbalance in semen pro- and antioxidant components after COVID-19.

Radiolysis Studies of Oxidation and Nitration of Tyrosine and Some Other Biological Targets by Peroxynitrite-Derived Radicals.

The widespread interest in free radicals in biology extends far beyond the effects of ionizing radiation, with recent attention largely focusing on reactions of free radicals derived from peroxynitrite (i.e., hydroxyl, nitrogen dioxide, and carbonate radicals). These radicals can easily be generated individually by reactions of radiolytically-produced radicals in aqueous solutions and their reactions can be monitored either in real time or by analysis of products. This review first describes the general principles of selective radical generation by radiolysis, the yields of individual species, the advantages and limitations of either pulsed or continuous radiolysis, and the quantitation of oxidizing power of radicals by electrode potentials. Some key reactions of peroxynitrite-derived radicals with potential biological targets are then discussed, including the characterization of reactions of tyrosine with a model alkoxyl radical, reactions of tyrosyl radicals with nitric oxide, and routes to nitrotyrosine formation. This is followed by a brief outline of studies involving the reactions of peroxynitrite-derived radicals with lipoic acid/dihydrolipoic acid, hydrogen sulphide, and the metal chelator desferrioxamine. For biological diagnostic probes such as 'spin traps' to be used with confidence, their reactivities with radical species have to be characterized, and the application of radiolysis methods in this context is also illustrated.

A Fluorescent Cage for Supramolecular Sensing of 3-Nitrotyrosine in Human Blood Serum.

3-Nitrotyrosine (NT) is generated by the action of peroxynitrite and other reactive nitrogen species (RNS), and as a consequence it is accumulated in inflammation-associated conditions. This is particularly relevant in kidney disease, where NT concentration in blood is considerably high. Therefore, NT is a crucial biomarker of renal damage, although it has been underestimated in clinical diagnosis due to the lack of an appropriate sensing method. Herein we report the first fluorescent supramolecular sensor for such a relevant compound: Fluorescence by rotational restriction of tetraphenylethenes (TPE) in a covalent cage is selectively quenched in human blood serum by 3-nitrotyrosine (NT) that binds to the cage with high affinity, allowing a limit of detection within the reported physiological concentrations of NT in chronic kidney disease.

Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase.

Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr-calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.

Changes in the Biomarkers of Oxidative/Nitrosative Stress and Endothelial Dysfunction Are Associated with Cardiovascular Risk in Periodontitis Patients.

Patients with cardiovascular disease (CVD) and periodontitis (PT) show shared risk factors as result of the altered molecular mechanisms associated with pathological conditions. The aim of our study was to evaluate if the plasma biomarkers associated with endothelial dysfunction may also be related to alterations in the inflammatory status in peripheral blood mononuclear cells (PBMC). Patients with PT, coronary heart disease (CHD), or both diseases as well as controls were enrolled. Plasma levels of coenzyme Q10 (CoQ10), 3-nitrotyrosine (NT), and asymmetric dimethylarginine (ADMA) were assessed using HPLC. mRNA levels of caspase-1 (CASP1), NLR family pyrin domain containing 3 (NLRP3), and tumor necrosis factor-α (TNF-α) in PBMC from the recruited subjects were quantified using real-time PCR. Patients with PT + CHD showed lower CoQ10 plasma levels and increased concentrations of NT in comparison to healthy subjects. ADMA levels were higher in CHD and PT + CHD patients compared to controls. Transcript levels of CASP1, NLRP3, and TNF-α were up-regulated in PBMC from all patient groups when compared to healthy subjects. Our results suggest a possible causal link between oxidative stress, high levels of NT and ADMA, and inflammasome activation, which may be involved in the endothelial inflammatory dysfunction leading to the pathogenesis and progression of CHD in PT patients.